Vaccines comprising aluminium adjuvants and histidine

ABSTRACT

To improve the stability of vaccines comprising aluminum salt(s), the invention uses the amino acid histidine. This can improve pH stability and adjuvant adsorption and can be reduce antigen hydrolysis. Histidine is preferably presen during adsorption to the aluminium salt(s). The antigen in the vaccine may be a protein or a saccharide and is preferably from  N. meningitidis.

TECHNICAL FIELD

This invention is in the field of vaccine formulation.

BACKGROUND ART

As well as containing antigenic substances, vaccines contain substancessuch as diluents, excipients, preservatives, stabilisers and buffers.Typically, vaccines also contain adjuvants i.e. a substance whichimproves the immune response raised in response to the vaccine antigen.

The adjuvants traditionally used in human vaccines have been aluminiumsalts such as aluminium hydroxide and aluminium phosphate. Many otherexperimental adjuvants are known and these are reviewed in, forinstance, reference 1. Adsorption to aluminium salts remains, however,the most common vaccine adjuvant formulation.

Although their use is widespread, aluminium salts may not always becompatible with particular antigens. It has been suggested, forinstance, that aluminium hydroxide may not be suitable for use inmultivalent vaccines including hepatitis B virus surface antigen [2] orfor use with the capsular polysaccharide from Haemophilus influenzae[3]. It has also been suggested that different antigens within the samevaccine formulation should be adsorbed to different aluminium salts [4]for compatibility reasons.

As well as antigen compatibility, it is necessary to consider vaccinestability when using aluminium salts. For instance, their capacity forprotein adsorption has been shown to drop over time at room temperature[5] and in response to autoclaving [6]. Alum salts may also causedifficulties in freeze drying [7]. Furthermore, it has been found thataluminium hydroxide can hydrolyse saccharide antigens [8], even at lowtemperatures and when the antigen is conjugated to a carrier protein,thus leading to reduced efficacy.

In general, these issues only arise when attention moves to formulatingan antigen for clinical use and may not be appreciated during initialresearch and development of the antigen itself.

It is an object of the invention to provide improvements in thestability of vaccines which include aluminium salts and, in particular,improvements in pH stability (buffering) and adjuvant adsorption atvarious temperatures and/or improvements in antigen stability (e.g.reduction in hydrolysis).

DISCLOSURE OF THE INVENTION

The invention is based on the surprising discovery that the amino acidhistidine enhances the stability of vaccines which include aluminiumsalt adjuvants. This has been found both for saccharide antigens and forprotein antigens.

The invention thus provides a composition comprising an antigen, analuminium salt and histidine.

The invention also provides a process for producing this composition,comprising the step of admixing an antigen, an aluminium salt andhistidine.

The Antigen

The antigen is preferably a protein antigen or a saccharide antigen(optionally conjugated). Preferred antigens are from bacteria, with thebacterial genus Neisseria (e.g. N. meningitidis) being particularlypreferred.

Specific bacterial antigens for use with the invention include:

-   a protein antigen from N, meningitidis serogroup B, such as those in    refs. 9 to 15, with protein ‘287’ (see below) and derivatives (e.g.    ‘ΔG287’) being particularly preferred,-   an outer-membrane vesicle (OMV) preparation from N. meningitidis    serogroup B, such as those disclosed in refs. 16, 17, 18, 19 etc.-   a saccharide antigen from N. meningitidis serogroup A, C, W135    and/or Y, such as the oligosaccharide disclosed in ref. 20 from    serogroup C [see also ref. 21].-   a saccharide antigen from Streptococcus pneumoniae [e.g. 22, 23,    24].-   an antigen from Bordetella pertussis, such as pertussis holotoxin    (PT) and filamentous haemagglutinin (FHA) from B. pertussis,    optionally also in combination with pertactin and/or agglutinogens 2    and 3 [e.g. refs. 25 & 26].-   a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter 3 of    ref. 27] e.g. the CRM₁₉₇ mutant [e.g. 28].-   a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of ref.    27].-   a protein antigen from Helicobacter pylori such as CagA [e.g. 29],    VacA [e.g. 29], NAP [e.g. 30], HopX [e.g. 31], HopY [e.g. 31] and/or    urease.-   a saccharide antigen from Haemophilus influenzae B [e.g. 21],    preferably oligosaccharide.-   an antigen from N. gonorrhoeae [e.g. 9, 10, 11].-   an antigen from Chlamydia pneumoniae [e.g. 32, 33, 34, 35, 36, 37,    38].-   an antigen from Chlamydia trachoniatis [e.g. 39].-   an antigen from Porphyromonas gingivalis [e.g. 40].-   an antigen from Moraxella catarrhalis [e.g. 41].-   an antigen from Streptococcus agalactiae (group B streptococcus)    [e.g. 42, 43].-   an antigen from Streptococcus pyogenes (group A streptococcus) [e.g.    43, 44, 45].-   an antigen from Staphylococcus aureus [e.g. 46].-   an antigen from Bacillus anthracis [e.g. 47, 48, 49].

Specific viral antigens for use with the invention include:

-   an antigen from hepatitis A virus, such as inactivated virus [e.g.    50, 51].-   an antigen from hepatitis B virus, such as the surface and/or core    antigens [e.g. 51, 52].-   an antigen from hepatitis C virus [e.g. 53].-   polio antigen(s) [e.g. 54, 55] such as IPV.-   rabies antigen(s) [e.g. 56] such as lyophilised inactivated virus    [e.g.57, RabAvert™].-   measles, mumps and/or rubella antigens [e.g. chapters 9, 10 & 1 of    ref. 27].-   influenza antigen(s) [e.g. chapter 19 of ref. 27], such as the    haemagglutinin and/or neuramimidase surface proteins.-   an antigen from a virus in the flaviviridae family (genus    flavivirus), such as from yellow fever virus, Japanese encephalitis    virus, four serotypes of Dengue viruses, tick-borne encephalitis    virus, West Nile virus.-   a pestivirus antigen, such as from classical porcine fever virus,    bovine viral diarrhoea virus, and/or border disease virus.-   a parvovirus antigen e.g. from parvovirus B19.

The composition may comprise one or more of these bacterial and viralantigens. The composition may comprise no viral antigens.

Other antigens which may be used include:

-   a prion protein (e.g. the CJD prion protein)-   an amyloid protein, such as a beta peptide [58]-   a cancer antigen, such as those listed in Table 1 of ref. 59 or in    tables 3 & 4 of ref. 60.

Where a saccharide or carbohydrate antigen is used, it is preferablyconjugated to a carrier protein in order to enhance mimunogenicity [e.g.refs. 61 to 70]. Preferred carrier proteins are bacterial toxins ortoxoids, such as diphtheria or tetanus toxoids. The CRM₁₉₇ diphtheriatoxoid is particularly preferred. Other suitable carrier proteinsinclude the N. meningitidis outer membrane protein [e.g. ref. 71],synthetic peptides [e.g. 72, 73], heat shock proteins [e.g. 74],pertussis proteins [e.g. 75, 76], protein D from H. influenzae [e.g.77], toxin A or B from C. difficile [e.g. 78], etc. Where a mixturecomprises capsular saccharides from both serogroups A and C, it ispreferred that the ratio (w/w) of MenA saccharide:MenC saccharide isgreater than 1 (e.g. 2:1, 3:1, 4:1, 5:1, 10:1 or higher). Saccharidesfrom different serogroups of N. meningitidis may be conjugated to thesame or different carrier proteins.

Any suitable conjugation reaction can be used, with any suitable linkerwhere necessary.

Toxic protein antigens may be detoxified where necessary (e.g.detoxification of pertussis toxin by chemical and/or genetic means[26]).

Human papilloma virus (HPV) virus-like particles (VLPs) are notpreferred antigens (cf. WO00/45841, WO00/57906, WO01/28585).

Where a diphtheria antigen is included in the composition it ispreferred also to include tetanus antigen and pertussis antigens.Similarly, where a tetanus antigen is included it is preferred also toinclude diphtheria and pertussis antigens. Similarly, where a pertussisantigen is included it is preferred also to include diphtheria andtetanus antigens. Whole cell pertussis antigen may be used.

Antigen is preferably adsorbed to the aluminium salt.

Where HBsAg is present, preferably it is either adsorbed to aluminiumhydroxyphosphate or is not adsorbed to any salt. Adsorption of HBsAg toan aluminium hydroxide is preferably avoided.

Where a H. influenzae saccharide antigen is present, preferably it iseither adsorbed to aluminium hydroxyphosphate or is not adsorbed to anysalt. Adsorption of Hib saccharides to an aluminium hydroxide ispreferably avoided.

Antigens in the composition will typically be present at a concentrationof at least 1 μg/ml each. In general, the concentration of any givenantigen will be sufficient to elicit an immune response against thatantigen.

As an alternative to using proteins antigens in the composition of theinvention, nucleic acid encoding the antigen may be used [e.g. refs. 79to 87]. Protein components of the compositions of the invention may thusbe replaced by nucleic acid (preferably DNA e.g. in the form of aplasmid) that encodes the protein.

The Aluminium Salt

The aluminium salt is preferably an aluminium hydroxide (e.g. aluminiumoxyhydroxide) or an aluminium phosphate (e.g. aluminium hydroxyphosphateor orthophosphate), but any other suitable salt may also be used (e.g.sulphate etc. [e.g. see chapters 8 & 9 of ref. 1]). The salt may takeany suitable form (e.g. gel, crystalline, amorphous etc.). Preferredsalts are (amorphous) hydroxyphosphates and (crystalline) oxyhydroxide(boehmite).

Hydroxyphosphates are obtained by precipitation and the reactionconditions and reactant concentrations during the precipitation reactioninfluence the degree of substitution of phosphate for hydroxyl in thesalt. Hydroxyphosphates generally have a PO₄/Al molar ratio between 0.3and 0.99, and preferred salts have a ratio between 0.8 and 0.95 (e.g.0.88±0.05). Hydroxyphosphates [Al(OH)_(x)(PO₄)_(y), wherein the sum ofthe valence of each anion times its mole fraction is −3] can bedistinguished from AlPO₄ by the presence of hydroxyl groups. Forexample, an IR spectrum band at 3146 cm⁻¹ (e.g. when heated to 200° C.)indicates the presence of structural hydroxyls.

Aluminium oxyhydroxide [AlO(OH)] can be distinguished from Al(OH)₃ by IRspectroscopy, in particular by the presence of an adsorption band at1070 cm⁻¹ and a strong shoulder at 3090-3100 cm⁻¹,

Mixtures of different aluminium salts may also be used. It is preferred,however, to use essentially a single salt e.g. where two salts are used,the ratio of one to the other is at least 5:1 by weight e.g. at least10:1, 100:1, 1000:1 etc.

The salt will generally be present such that the concentration of Al³⁺is at least 1 μg/ml (e.g. at least 10 μg/ml, at least 100 μg/ml etc.).

The use of histidine in combination with an aluminium phosphate(particularly a hydroxyphosphate) is particularly advantageous foracidic antigens.

The Histidine

Histidine is a standard amino acid and is readily available for use withthe invention. As it is inherently biocompatible, it is safe, and thusadvantageous as an component in vaccines.

The concentration of histidine in the composition will typically be atleast 1 μm and at most 1M. The concentration is preferably at least 1 mM(e.g. at least 2 mM, 3 mM, 4 mM, 5 mM etc.) and is preferably at most250 mM (e.g. at most 200 mM, 150 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM,50 mM, 40 mM, 30 mM, 20 mM, 10 mM etc.). More preferably theconcentration of histidine in the composition is between 2 mM and 10 mM(e.g. between 5 mM and 8 mM) and, most preferably, it is about 5 mM.

The histidine is preferably L-histidine.

The histidine preferably acts as a buffer. Histidine buffers are wellknown to the skilled person.

Accordingly, the histidine may be ionised within the composition of theinvention.

The composition preferably has enhanced pH stability and/or reducedantigen hydrolysis when compared to an equivalent composition in whichhistidine buffer system is either replaced with a sodium phosphatebuffer system or in which no buffer system is included. Reducedhydrolysis may be a consequence of enhanced pH stability.

Histidine may be added to the composition in the form of the amino aciditself or in the form of a salt. A typical histidine salt is themonohydrochloride monohydrate.

It will be appreciated that references to histidine in the compositionsof the invention refers to ‘free’ histidine rather than to any histidineresidues which may be part of a polypeptide (e.g. the antigen) withinthe composition.

Further Characteristics of the Composition

The composition is preferably in liquid form, but it may be lyophilised(cf. WO01/41800).

The composition may also comprise a sodium salt e.g. sodium phosphate orsodium chloride. The concentration of the sodium salt is preferably atleast 1 mM (e.g. at least 2 mM, 3 mM, 4 mM, 5 mM etc.) and is preferablyat most 10 mM (e.g. at most 10 mM, 9 mM, 8 mM, 7 mM etc.). Morepreferably the concentration of sodium salt in the composition isbetween 1 mM and 5 mM (e.g. between 2 mM and 3 mM) and, most preferably,it is about 2.5 mM.

A particular advantage of the invention is that it allows good controlof pH and adsorption in vaccines which contain high concentrations offree phosphate ions, which ions may be unavoidable in the vaccine e.g.due to exchange with phosphates in the adjuvant, or due to residualphosphate buffer. Where residual phosphate ions are present at between 3and 5 mM, for example, pH is difficult to control between 6.0 and 7.0,and some antigens tend to desorb from adjuvants, but the addition of 5to 10 mM histidine pH and adsorption to be controlled, including duringstorage at elevated temperatures.

The molar ratio of histidine to free phosphate is preferably at least1.25:1 e.g. 1.5:1, 1.75:1, 2:1, 2.25:1, 2.5:1, 3:1, 4:1 etc.

The pH of the composition is preferably between 6 and 7 (e.g. betweem6.3 and 7.0). The pH may be maintained by the use of a buffer. This willtypically be achieved inherently by the histidine in the composition.

The composition will not, in general, contain: serum (e.g. fetal calfserum etc.) or other such components used in cell culture; host cell DNAat a level of greater than 100 pg/dose for antigens purified from cellculture; living cells.

The composition will generally be sterile and/or pyrogen-free.

The composition may comprise a detergent (e.g. a Tween, such as Tween80) in order to minimise adsorption of antigens to containers.

The composition preferably does not comprise a preservative. Where apreservative is present, mercurial preservatives (e.g. thimerosal) maybe used (cf. WO98/34594). Preservatives which may be present or absentare 2-phenoxy-ethanol, methyl parabens, propyl parabens and benzylalcohol (or mixtures thereof).

Immunogenic Compositions and Medicaments

The composition of the invention is typically a vaccine composition.

The invention also provides a composition of the invention for use as amedicament. The medicament is preferably able to raise an immuneresponse in a mammal against the antigen (i.e. it is an immunogeniccomposition) and is more preferably a vaccine.

The invention also provides the use of a composition of the invention inthe manufacture of a medicament for raising an immune response in amammal against the antigen. The medicament is preferably a vaccine.

The invention also provides a method for raising an immune response in amammal comprising the step of administering an effective amount of acomposition of the invention. The immune response is preferablyprotective. The method may raise a booster response.

The mammal is preferably a human, and most preferably a child.

These uses and methods are preferably for the prevention and/ortreatment of a disease caused by a Neisseria (e.g. meningitis,septicaemia; gonorrhoea etc.), by H. influenzae (e.g. otitis media,bronchitis, pneumonia, cellulitis, pericarditis, meningitis etc.) or bypneumococcus (e.g. meningitis, sepsis, pneumonia etc). The preventionand/or treatment of bacterial meningitis is thus preferred.

Vaccines according to the invention may either be prophylactic (i.e. toprevent infection) or therapeutic (i.e. to treat disease afterinfection), but will typically be prophylactic.

Further Components of the Composition

The composition of the invention will typically, in addition to thecomponents mentioned above, comprise one or more ‘pharmaceuticallyacceptable carriers’, which include any carrier that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition. Suitable carriers are typically large, slowlymetabolised macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,trehalose (WO00/56365) and lipid aggregates (such as oil droplets orliposomes). Such carriers are well known to those of ordinary skill inthe art. The vaccines may also contain diluents, such as water, saline,glycerol, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, and the like, may bepresent. A thorough discussion of pharmaceutically acceptable excipientsis available in Remington's Pharmaceutical Sciences [e.g. ref. 88].

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of antigen, as well as any other of the above-mentionedcomponents, as needed. By ‘immunologically effective amount’, it ismeant that the administration of that amount to an individual, either ina single dose or as part of a series, is effective for treatment orprevention. This amount varies depending upon the health and physicalcondition of the individual to be treated, age, the taxonomic group ofindividual to be treated (e.g. non-human primate, primate, etc.), thecapacity of the individual's immune system to synthesise antibodies, thedegree of protection desired, the formulation of the vaccine, thetreating doctor's assessment of the medical situation, and otherrelevant factors. It is expected that the amount will fall in arelatively broad range that can be determined through routine trials.Dosage treatment may be a single dose schedule or a multiple doseschedule (e.g. including booster doses). The vaccine may be administeredin conjunction with other immunoregulatory agents.

The vaccine may be administered in conjunction with otherimmunoregulatory agents.

The vaccine may include an adjuvant in addition to the aluminium salt.Preferred adjuvants to enhance effectiveness of the composition include,but are not limited to: (1) oil-in-water emulsion formulations (with orwithout other specific immunostimulating agents such as muramyl peptides(see below) or bacterial cell wall components), such as for example (a)MF59™ (WO90/14837; Chapter 10 in ref. 1), containing 5% Squalene, 0.5%Tween 80, and 0.5% Span 85 (optionally containing MTP-PE) formulatedinto submicron particles using a microfluidizer, (b) SAF, containing 10%Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDPeither microfluidized into a submicron emulsion or vortexed to generatea larger particle size emulsion, and (c) Ribi™ adjuvant system (RAS),(Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween80, and one or more bacterial cell wall components from the groupconsisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (2) saponinadjuvants, such as QS21 or Stimulon™ (Cambridge Bioscience, Worcester,Mass.) may be used or particles generated therefrom such as ISCOMs(immunostimulating complexes), which ISCOMS may be devoid of additionaldetergent e.g. WO00/07621; (3) Complete Freund's Adjuvant (CFA) andIncomplete Freund's Adjuvant (IFA); (4) cytokines, such as interleukins(e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO99/44636), etc.),interferons (e.g. gamma interferon), macrophage colony stimulatingfactor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryllipid A (MPL) or 3-O-deacylated MPL (3dMPL) e.g. GB-2220221,EP-A-0689454; (6) combinations of 3dMPL with, for example, QS21 and/oroil-in-water emulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231;(7) oligonucleotides comprising CpG motifs [Krieg Vaccine 2000, 19,618-622; Krieg Curr opin Mol Ther 2001 3:15-24; Roman et al., Nat. Med.,1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Daviset al., J. Immunol., 1998, 160, 870-876; Chu et al., J. Exp. Med., 1997,186, 1623-1631; Lipford et al., Eur. J. Immunol., 1997, 27, 2340-2344;Moldoveanu et al., Vaccine, 1988, 16, 1216-1224, Krieg et al., Nature,1995, 374, 546-549; Klinman et al., PNAS USA, 1996, 93, 2879-2883;Ballas et al., J. Immunol., 1996, 157, 1840-1845; Cowdery et al., J.Immunol., 1996, 156, 4570-4575; Halpern et al., Cell. Immunol., 1996,167, 72-78; Yamamoto et al., Jpn. J. Cancer Res., 1988, 79, 866-873;Stacey et al., J. Iimmunol., 1996, 157, 2116-2122; Messina et al., J.Immunol., 1991, 147, 1759-1764; Yi et al., J. Immunol., 1996, 157,4918-4925; Yi et al., J. Immunol., 1996, 157, 5394-5402; Yi et al., J.Iiizmuolno., 1998, 160, 4755-4761; and Yi et al., J. Immunol., 1998,160, 5898-5906; International patent applications WO96/02555,WO98/16247, WO98/18810, WO98/40100, WO98/55495, WO98/37919 andWO98/52581] i.e. containing at least one CG dinucleotide, with5-methylcytosine optionally being used in place of cytosine; (8) apolyoxyethylene ether or a polyoxyethylene ester e.g. WO99/52549; (9) apolyoxyethylene sorbitan ester surfactant in combination with anoctoxynol (e.g. WO01/21207) or a polyoxyethylene alkyl ether or estersurfactant in combination with at least one additional non-ionicsurfactant such as an octoxynol (e.g. WO01/21152); (10) animmunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) and asaponin e.g. WO00/62800; (11) an immunostimulant and a particle of metalsalt e.g. WO00/23105; (12) a saponin and an oil-in-water emulsion e.g.WO99/11241; (13) a saponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol)e.g. WO98/57659; (14) chitosan; (15) cholera toxin or E. coli heatlabile toxin, or detoxified mutants thereof [89]; (16) microparticles ofpoly(α-hydroxy)acids, such as PLG; (17) other substances that act asimmunostimulating agents to enhance the efficacy of the composition.

Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetylnormuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), etc.

Once formulated, the compositions of the invention can be administereddirectly to the subject. The subjects to be treated can be animals; inparticular, human subjects can be treated. The vaccines are particularlyuseful for vaccinating children and teenagers.

Typically, the immunogenic compositions are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared. The preparation also may be emulsified or encapsulatedin liposomes for enhanced adjuvant effect. Direct delivery of thecompositions will generally be parenteral (e.g. by injection, eithersubcutaneously, intraperitoneally, intravenously or intramuscularly ordelivered to the interstitial space of a tissue). The compositions canalso be administered into a lesion. Other modes of administrationinclude oral and pulmonary administration, suppositories, andtransdermal or transcutaneous applications (e.g. see WO98/20734),needles, and hyposprays. Dosage treatment may be a single dose scheduleor a multiple dose schedule (e.g. including booster doses).

The Step of Admixing Antigen, Aluminium Salt and Histidine

To make compositions of the invention, antigen, aluminium salt andhistidine must be combined. It is preferred that, when the antigen andaluminium salt are mixed, the histidine should be present. Histidine isthus present during adsorption to the aluminium salt. This compares withadding histidine to an antigen/aluminium salt combination which alreadyexists i.e. the histidine in the process is not simply added as a bufferafter antigen and aluminium salt have interacted, but instead it ispresent during their interaction.

In the process of the invention, therefore, antigen is preferablyadmixed with a histidine/aluminium salt mixture. The process of theinvention may therefore comprise the following steps: (a) preparing amixture of the aluminium salt and the histidine; and (b) admixing theantigen with said mixture. The mixture of (a) is preferably aqueous andmay be prepared in aqueous conditions or may be a dried mixture which isre-hydrated prior to use.

Once one or more antigens has been adsorbed to an aluminium salt in thepresence of histidine, the mixture may be combined with other antigense.g. combined with existing diphtheria, tetanus, pertussis, polio orhepatitis B virus compositions.

Definitions

The term “comprising” means “including” as well as “consisting” e.g. acomposition “comprising” X may consist exclusively of X or may includesomething additional e.g. X+Y.

The term “about” in relation to a numerical value x means, for example,x±10%.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows SDS-PAGE analysis of antigenic compositions followingcentrifugation. Lane 1 includes MW markers (220, 97, 66, 46, 30, 21, 14kDa). OMV antigen (2 μkg) was used in lane 2; ΔG287 antigen was used inlanes 3 (10 μg) and 4 (0.5 μg). The antigen used in lanes 5 and 6combination of OMV (50 μg/ml) and ΔG287 (100 μg/ml) with 1 mg/mlaluminium oxyhydroxide; the lane 5 composition included 10 mM sodiumphosphate (PBS), whereas the lane 6 composition included 5 mM histidinein saline solution.

FIG. 2 also shows SDS-PAGE analysis of antigenic compositions followingcentrifugation. Lane 1 includes the same MW markers as FIG. 1. OMVantigen (2.5 μg) was used in lane 2; ΔG287 antigen was used in lanes 3(2 μg) and 4 (0.5 μg). The antigen used in lanes 5, 6 and 7 was acombination of OMV (50 μg/ml) and ΔG287 (100 μg/ml) with 1 mg/mlaluminium oxyhydroxide in saline solution (pH 6.5); the lane 5composition included 2.5 mM sodium phosphate, the lane 6 compositionincluded 5 mM histidine, and the lane 7 composition included 10 mMhistidine.

FIG. 3 also shows SDS-PAGE analysis of antigenic compositions followingcentrifugation. Lane 1 includes the same MW markers as FIG. 1. OMVantigen (2 μkg) was used in lane 2; ΔG287 antigen was used in lanes 3 (2μg) and 4 (0.5 μg). The antigen used in lanes 5 and 6 was a combinationof OMV (50 μg/ml) and ΔG287 (100 μg/ml) with 3.3 mg/ml aluminiumoxyhydroxide in saline solution (pH 6.5); the lane 5 compositionincluded 2.5 mM sodium phosphate (PBS), whereas the lane 6 compositionincluded 5 mM histidine in saline solution.

FIG. 4 shows the pH stability of vaccine formulations at 4° C. Filledsymbols represent vaccines buffered with 5 mM histidine; open symbolsrepresent vaccines buffered with 2.5 mM sodium phosphate. The initial pHwas 6.0 (diamond), 6.5 (square) or 7.0 (triangle).

FIG. 5 shows the same at 37° C.

FIG. 6 shows a SDS-PAGE gel for various antigens. Lane 1 contains MWmarkers. Lanes 2 to 6 contain markers: (2) ΔG287-953; (3) 961c; (4)936-741; (5) New Zealand OMVs; and (6 Norwegian OMVs. Lanes 7 to 10 showsupernatants of centrifuged histidine formulations of the inventionafter 1 month storage at 2-8° C.: (7) ΔG287-953; (8)961c+936-741+ΔG287-953; (9) 961c+936-741+ΔG287-953+OMV_(NZ); (10)961c+936-741+ΔG287-953+OMV_(Norway).

FIG. 7 shows the same as FIG. 6, but lanes 7-10 are after storage at36-38° C.

FIG. 8 shows a SDS-PAGE gel for various antigens. Lane 1 contains MWmarkers. Lanes 2 to 5 contain markers: (2) 961c; (3) 936-741; (4) NewZealand OMVs; and (5) Norwegian OMVs. Lanes 6 to 9 show supernatants ofcentrifuged histidine formulations of the invention after 1 monthstorage at 2-8° C.: (6) 961c; (7) 936-741; (8) OMV_(NZ); (9)OMV_(Norway).

FIG. 9 shows the same as FIG. 8, but lanes 6-9 are after storage at36-38° C.

FIG. 10 shows a SDS-PAGE gel for New Zealand OMVs. Lane 1 contains MWmarkers. Lanes 2, 3, 6 & 7 contain OMV markers stored at either 2-8° C.(lanes 2 & 3) or 36-38° C. (lanes 6 & 7), present at either 2 μg lanes 2& 6) or 1 μg (lanes 3 & 7). Lanes 4, 5, 8 & 9 show OMVs in histidineformulations of the invention after 30 days storage at either 2-8° C.(lanes 4 & 5) or 36-38° C. (lanes 8 & 9). Lanes 4 & 8 show supernatantof centrifuged OMVs, whereas lanes 5 & 9 show pellets.

MODES FOR CARRYING OUT THE INVENTION EXAMPLE 1 pH Stability andAdsorption of Menigococcal B ‘287’ Antigen

Reference 11 discloses a protein antigen named ‘287’ from N.meningitidis serogroup B. Reference 90 discloses a form of this antigen(‘ΔG287’) which is truncated to remove the N-terminal amino acids up toand including its hexaglycine region. 287 and ΔG287 are both able toelicit a protective immune response in mice. References 16 to 19disclose OMV antigens from N. meningitidis serogroup B. These OMVs arealso able to elicit a protective immune response in mice.

These two antigens were formulated by adsorption to aluminiumoxyhydroxide adjuvant. Two adjuvant concentrations (1 mg/ml and 3.3mg/ml) were tested.

Immunisation studies in mice showed that vaccine immunogenicity islinked to the level of adsorption of the antigens to the adjuvant. Toassess adsorption levels, samples of the final formulations werecentrifuged at 1300 rpm for 10 minutes and the supernatant was analysedby SDS-PAGE in order to detect the presence of non-adsorbed antigen. Therelevant protein standards at an appropriate concentration were loadedadjacent for quantitative comparison.

In order to maintain a stable physiological pH at 4° C. and 37° C. overa period of 4 weeks using sodium phosphate buffer it was found that thecomposition requires 10 mM sodium phosphate. At this level, however,adsorption of ΔG287 was only 50% (FIG. 1, lane 5). 100% adsorption couldbe maintained at 2.5 mM sodium phosphate (Lanes 5 of FIGS. 2 & 3), butthis composition does not have a stable pH at either 4° C. or 37° C.

It was therefore necessary to find an alternative buffer system whichwould maintain pH stability without decreasing adsorption.

Adsorption was 95-100% using 5 mM histidine (Lanes 6 of FIGS. 1, 2 & 3)and also using 10 mM histidine (FIG. 2, lane 7). In terms of adsorption,therefore, 5 mM or 10 mM histidine was equivalent to 2.5 mM sodiumphosphate in the presence of either 1 mg/ml (FIGS. 1 & 2) or 3.3 mg/ml(FIG. 3) aluminium oxyhydroxide.

In order to define the pH range in which the vaccine compositions arestable, three starting pH values were chosen (pH 6.0, 6.5 and 7.0) andpH stability was monitored over four weeks in the presence of either 2.5mM sodium phosphate or 5 mM histidine. Stability was monitored at both4° C. and 37° C.

The antigen in all vaccines was a combination of ΔG287 (100 μg/ml) andOMV (50 μg/ml) adjuvanted with 3.3 mg/ml aluminium oxyhydroxide.

FIG. 4 shows pH stability at 4° C. and FIG. 5 shows pH stability at 37°C. [NB—due to bacterial contamination, no measurement of the pH 6.0histidine-buffered vaccine was possible at 4 weeks].

At both temperatures the pH tended to increase over time with 2.5 mMsodium phosphate buffer but was stable in the presence of 5 mM histidinebuffer.

A In comparison with sodium phosphate buffer, therefore, the use ofhistidine offers pH stability over time without reducing adsorption.

EXAMPLE 2 Adsorption of Meningococcal C Saccharide Antigen

Saccharide conjugates tend to degrade by hydrolysis [7,8] when presentin solution (‘liquid’ vaccines). Conjugates can be lyophilised to avoidthis [7], but this requires adjuvant to be added at the point ofreconstitution. It would be preferable to have a liquid form of thevaccine in which the saccharide is not subject to hydrolyticdegradation.

This was investigated for a conjugate of meningococcus serogroup Coligosaccharide on CRM₁₉₇ carrier protein [20]. CRM₁₉₇ is acidic andthus does not completely adsorb to negatively charged aluminiumphosphates. Histidine, however, is positively charged and it was thoughtthat this might be able to mask the negative charge. Histidine bufferwas thus tested with the aim of improving adsorption of MenC-CRM₁₉₇ toaluminium hydroxyphosphate.

Antigen adsorption was evaluated in the presence and absence ofhistidine buffer by measuring protein concentration in the vaccinesupernatant using the BCA protein assay, after centrifugation toseparate the adjuvant pellet. The vaccines were formulated as 20 μg/mloligosaccharide and 454 μg/ml CRM₁₉₇ protein. Results were as follows:

Antigen Adjuvant [Histidine] (mM) Protein (μg/ml) MenC-CRM₁₉₇Hydroxyphosphate 0 42.4 Al³⁺ = 0.6 mg/ml 5 28.6 10 21.7

Antigen adsorption thus improves when histidine is present in theformulation: adsorption is about 6% in the absence of histidine; 5 mMhistidine increases this to 36%; 10 mM histidine increases adsorption toalmost 52%.

Histidine is thus a useful additive for improving the adsorption ofantigens to aluminium hydroxyphosphate.

EXAMPLE 3 Adsorption of Meningococcal B NadA Antigen

NadA (Neisserial adhesin A) from serogroup B N. meningitidis isdisclosed as protein ‘961’ in ref. 11 (SEQ IDs 2943 & 2944) and as‘NMB1994’ in ref. 13 (see also GenBank accession numbers 11352904 &7227256). Allelic forms of NadA are disclosed in reference 91. Preferredforms of NadA lack the C-terminus anchor domain (‘961c’).

961c (100 μg/ml) was adsorbed onto aluminium oxyhydroxide (3 mg/ml) inthe presence of 10 mM histidine buffer, pH 6.5. After 4 weeks of storageat either 2-8° C. or at 36-38° C., the antigen remained 100% adsorbed(FIGS. 8 & 9, lane 6). The pH of the composition was 6.44 at time zeroand after 4 weeks of storage rose very slightly to 6.48 (2-8° C.) or6.47 (36-38° C.).

EXAMPLE 4 Adsorption of Meningococcal B Hybrid Antigens

References 92 & 93 disclose hybrid expression of meningococcal Bantigens. One such hybrid is ‘ΔG287_(nz)-953’ and another is ‘936-741’.These two hybrids (100 μg/ml) were each adsorbed onto aluminiumoxyhydroxide (3 mg/ml) in the presence of 10 mM histidine buffer, pH6.3. After 4 weeks of storage at either 2-8° C. or at 36-38° C.,‘ΔG287_(nz)-953’ remained 100% adsorbed (FIGS. 6 & 7, lane 7), with pHrising slightly from 6.44 to 6.52 (2-8° C.) or 6.53 (36-38° C.).‘936-741’ remained 100% adsorbed at 36-38° C. (FIG. 9, lane 7) but was˜99% adsorbed at 2-8° C. (FIG. 8, lane 7), with pH rising slightly from6.33 to 6.37 (2-8° C.) or 6.38 (36-38° C.).

EXAMPLE 5 Adsorption of Menizzgococcal OMVs

As mentioned above, OMV vaccines from meningococcus B are well known.OMVs were prepared from the Norwegian strain of meningococcus B or froma New Zealand strain (394/98). These two OMV preparations (50 μg/ml)were adsorbed onto aluminium oxyhydroxide (3 mg/ml) in the presence of10 mM histidine buffer, pH 6.5. After 4 weeks of storage at either 2-8°C. or at 36-38° C., both OMV preparations remained 100% adsorbed (FIGS.8 & 9, lanes 8 & 9). For the Norwegian OMVs, pH rose slightly from 6.39to 6.42 over 4 weeks at both storage temperatures. For the New ZealandOMVs, pH rose slightly from 6.40 to 6.42 (2-8° C.) or 6.43 (36-38° C.).

New Zealand OMVs were alternatively formulated with 5 mM histidine.Starting with pure water, the aluminium oxyhydroxide was added, followedby histidine, with 10 minutes mixing. The OMVs were then added and mixedfor 15 minutes. NaCl was then added followed by 10 minutes furthermixing. The final composition was 3.3 mg/ml aluminium oxyhydroxide, 7.5mM NaCl, 5 mM histidine, 100 μg/ml OMV, pH 6.42.

During storage at either 2-8° C. or 36-36° C., pH and OMV adsorptionvaried as follows:

pH % Adsorption 2-8° C. 36-38° C. 2-8° C. 36-38° C. Time zero 6.42 6.42100 100 15 days 6.36 6.37 100 100 30 days 6.35 6.34 100 100

A comparison of lanes 4 & 5 (2-8° C.) or lanes 8 & 9 (36-38° C.) in FIG.10 shows that OMVs remain adsorbed after 1 month of storage.

EXAMPLE 6 Adsorption of Mixtures of Meningococcal OMVs and ProteinAntigens

961c, ΔG287_(nz)-953 and 936-741 were mixed at 100 μg/ml of each antigenand the mixture was adsorbed onto aluminium oxyhydroxide (3 mg/ml) inthe presence of 10 mM histidine buffer, pH 6.3. In two furtherformulations, OMVs were included (50 μg/ml) from either Norwegian or NewZealand strain meningococcus B.

All antigens in the three mixtures (FIGS. 6 & 7, lanes 8-10) showed 100%adsorption after 4 weeks of storage at either 2-8° C. or at 36-38° C.,except for 936-741 which was ˜96% adsorbed in all three mixtures at 2-8°C. and ˜99% adsorbed at 36-38° C. The pH of each of the three mixturesrose slightly from 6.53 at time zero to 6.62 after 4 weeks at 2-8° C. At36-38° C., the pH of three mixtures rose to 6.71±0.02.

The individual antigens brought residual phosphate ions into the mixturefrom their own PBS. Phosphate ions were sometimes present at between 3and 5 mM in the combined antigen mixture. In the presence of these highconcentrations of residual phosphate buffer, it was difficult tostabilise pH within 6.0 to 7.0, even with 5 mM histidine. When histidinewas increased to 10 mM, however, pH was stabilised. Furthermore, theantigens remained adsorbed even after 1 month of storage at either 2-8°C. or at 36-38° C.

EXAMPLE 7 Adsorption of Meningococcal A Saccharide Antigen

Reference 94 discloses CRM₁₉₇ conjugates of capsular oligosaccharidefrom serogroup A meningococcus. The conjugates are not fully stable andare therefore prepared in lyophilised form, ready for re-constitution atthe time of administration. The lyophilised form was prepared to havecomponents which give the following composition after reconstitutioninto a unit dose:

Component Concentration CRM-MenA 20 μg saccharide/ml Potassium phosphatebuffer  5 mM Mannitol 15 mg/mlThis composition has no adjuvant, so an adjuvant was prepared for itsreconstitution:

Component Concentration Aluminium oxyhydroxide 0.68 mg Al³⁺/ml Histidinebuffer   10 mM Sodium chloride   9 mg/ml Tween 80 0.005% PH 7.2 ± 0.05 *amorphous hydroxyphosphate, PO₄/Al molar ratio between 0.84 and 0.92

EXAMPLE 8 Adsorption of Meningococcal C, W135 and Y Saccharide Antigens

Reference 94 discloses CRM₁₉₇ conjugates of capsular oligosaccharidesfrom meningococcus serogroups C, W135 and Y. A trivalent mixture of thethree conjugates either adsorbed onto an aluminium oxyhydroxide adjuvant(2 mg/ml) or an aluminium hydroxyphosphate adjuvant (0.6 mg/ml Al³⁺) wasprepared. The compositions of the two trivalent mixtures were asfollows:

Component Concentration Concentration Aluminium 0.68 mg Al³⁺/ml —oxyhydroxide Aluminium —  0.6 mg Al³⁺/ml hydroxyphosphate* CRM-MenC   20μg saccharide/ml   20 μg saccharide/ml CRM-MenY   20 μg saccharide/ml  20 μg saccharide/ml CRM-MenW135   20 μg saccharide/ml   20 μgsaccharide/ml Sodium phosphate buffer —   10 mM Histidine buffer   10 mM— Sodium chloride   9 mg/ml   9 mg/ml Tween 80 0.005% 0.005% *amorphoushydroxyphosphate, PO₄/Al molar ratio between 0.84 and 0.92

For the oxyhydroxide/histidine formulation, stability of the saccharidecomponents either in the bulk mixture or after packaging into vials wasas follows:

Stored at 2-8° C. Stored at 36-38° C. Free Free Free Free Timesaccharide saccharide saccharide saccharide (days) (μg/ml) % (μg/ml) %MenC bulk 0 <1.2 <6 <1.2 <6 15 <1.2 <6 <1.2 <6 30 <1.2 <6 <1.2 <6 MenCvials 0 <1.2 <6 <1.2 <6 15 <1.2 <6 <1.2 <6 30 <1.2 <6 1.3 6.6 MenW135bulk 0 2.5 12.5 2.5 12.5 15 2.3 11.4 3.4 16.8 30 2.3 11.5 3.5 17.3MenW135 vials 0 2.1 10.6 2.1 10.6 15 2.3 11.7 2.7 13.3 30 20. 10.2 3.316.3 MenY bulk 0 1.7 8.3 1.7 8.3 15 <1.3 <6.3 2.0 10.2 30 1.3 6.3 2.412.2 MenY vials 0 1.4 7.1 1.4 7.1 15 1.5 7.6 2.1 10.7 30 1.3 6.3 2.914.3

Free saccharide levels are thus stable for at least 1 month at 2-8° C.,before and after packaging.

Under thermal stress conditions, small increases in free saccharide areseen over time for MenW135 and MenY, but MenC remains stable.

Over the 30 days, pH in vials and bulk was stable at 7.15±0.05 at bothstorage temperatures.

EXAMPLE 9 Adsorption of Menizgococcal A, C, W135 and Y SaccharideAntigens

The two trivalent liquid compositions of example 8 were diluted and 0.5ml used to reconstitute the lyophilised MenA conjugate of example 7. Theresulting tetravalent mixture was administered to ten Balb/c mice(female 6-8 weeks old) per group by subcutaneous injection at day 0 and28. The mixture contained 2 μg of each saccharide conjugate per dose,which represents ⅕ of the single human dose (SHD). Controls were salineor unconjugated homologous polysaccharides. Bleedings were performedbefore immunization and then at day 42, with sera stored at −70° C.

All the conjugates used were safe and immunogenic in the animals. GMTpost-II ELISA titres (with 95% confidence intervals) were as follows:

Vaccine Adjuvant A Y W135 C MenA (lyophilised Hydroxyphosphate 172 — — —and resuspended) (69-439) Oxyhydroxide 619 — — — (419-906) MenYHydroxyphosphate — 328 — — (147-731) Oxyhydroxide — 452 — — (344-593)MenW Hydroxyphosphate — —  80 — (28-225) Oxyhydroxide — — 277 —(185-411) MenC Hydroxyphosphate — — — 317 (152-659) Oxyhydroxide — — —723 (615-851) MenA (lyophilized) + MenC, Hydroxyphosphate  32 397  99114 W135, Y (15-68) (252-627) (35-288) (53-246) Oxyhydroxide 206 141 139163 (112-372) (97-205) (76-251) (122-218)

Typically, therefore, titres are higher in the aluminiumoxyhydroxide+histidine groups. Serum bactericidal titres were alsogenerally better in the aluminium oxyhydroxide+histidine groups.

In parallel experiments, mice were immunised as described above but thevaccine compositions contained different ratios of the variousoligosaccharide conjugates. Lyophilised MenA oligo-conjugate was used inall experiments. ELISA titreswere as follows:

Antigen quantity (μg/dose) Aluminium GMT ELISA (95% confidence interval)A C W135 Y adjuvant A C W135 Y 4 2 2 2 Hydroxyphosphate 177 367 239 239(107-291) (263-510) (135-424) (184-311) 4 2 2 2 Oxyhydroxide 390 494 338158 (313-486) (345-706) (266-430) (96-260) 2 2 2 2 Hydroxyphosphate 132582 143 247 (59-296) (268-1155) (75-272) (152-400) 2 2 2 2 Oxyhydroxide337 569 171 100 (239-476) (462-679) (117-251) (59-169)

A second set of experiments was performed using a dosage of 2 μg/mlsaccharide for MenA and MenC, half that dosage for MenY, and a quarterdosage for MenW135. ELISA titres were as follows:

Antigen quantity (μg/dose) Aluminium GMT ELISA (95% confidence interval)A C W135 Y adjuvant A C W135 Y 2 2 2 2 Hydroxyphosphate  32 114  99 397(15-68) (53-246) (35-288) (252-627) Oxyhydroxide 206 163 139 141(112-372) (122-218) (76-251) (97-205) 2 2 1 0.5 Hydroxyphosphate  96 238 42 315 (49-187) (101-561) (20-89) (114-867) Oxyhydroxide 293 267  83244 (144-597) (158-451) (43-163) (152-392)

At least for serogroups A, C and W135, therefore, theoxyhydroxide+histidine formulation generally gives better titres thanhydroxyphosphate at these different antigen ratios.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES (THE CONTENTS OF WHICH ARE HEREBY INCORPORATED BY REFERENCE)

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1. A composition comprising an antigen, an aluminium salt and histidine,said composition further comprising at least about 2.5 mM of freephosphate, wherein said composition has a histidine to free phosphatemolar ratio of at least 1.25:1.
 2. The composition of claim 1, whereinthe antigen is a protein antigen or a saccharide antigen.
 3. Thecomposition of claim 1 or claim 2, wherein the antigen is a bacterialantigen selected from the group consisting of: a protein antigen from N.meningitidis; an outer-membrane vesicle (OMV) preparation from N.meningitidis; a saccharide antigen from N. meningitidis; a saccharideantigen from Streptococcus pnemnoniae; an antigen from Bordetellapertussis; a diphtheria antigen; a tetanus antigen; a protein antigenfrom Helicobacter pylori; a saccharide antigen from Haemophilusinfluenzae; an antigen from N. gonorrhoeae; an antigen from Chlamydiapneumoniae; an antigen from Chiamydia trachomatis; an antigen fromPorphyromonas gingivalis; an antigen from Moraxella catarrhalis; anantigen from Streptococcus agalactiae; an antigen from Streptococcuspyogenes; and an antigen from Staphylococcus aureus.
 4. The compositionof claim 3, wherein the antigen is from Neisseria menningitidisserogroup B.
 5. The composition of claim 2, wherein the saccharideantigen is a conjugated oligosaccharide antigen.
 6. The composition ofclaim 1, wherein the antigen is adsorbed to the aluminium salt.
 7. Thecomposition of claim 6, wherein the aluminium salt is a hydroxide or aphosphate, or a mixture of two or more of said salts.
 8. The compositionof claim 1, wherein aluminium salt is aluminium hydroxyphosphate and theantigen is an acidic antigen.
 9. The composition of claim 1, wherein theconcentration of histidine is between about 5 mM and about 10 mM. 10.The composition of claim 1, further comprising a sodium salt.
 11. Thecomposition of claim 10, wherein the concentration of the sodium salt isbetween about 2.5 mM and about 5 mM.
 12. The composition of claim 1,wherein the pH of the composition is between 6 and
 7. 13. Thecomposition of claim 1, further comprising a pharmaceutically acceptablecarrier.
 14. The composition of claim 1, comprising more than oneantigen.
 15. The composition of claim 14, wherein more than one of theantigens is adsorbed to an aluminium salt.
 16. The composition of claim14 or claim 15, comprising 2, 3, 4, 5, 6 or 7 of the following: anantigen from Bordetella pertussis; a diphtheria antigen; a tetanusantigen; an antigen from hepatitis B virus; a saccharide antigen fromHaemophilus influenzae; inactivated polio virus; and a saccharideantigen from N. meningitidis serogroup C.
 17. A method for raising animmune response in a mammal comprising the step of administering aneffective amount of the composition of claim
 1. 18. The method of claim17, wherein the mammal is a human.
 19. A process for producing anantigenic composition having improved stability, the process comprisingadmixing one or more antigens, an aluminium salt and histidine, whereinhistidine is present during adsorption of the one or more antigens tothe aluminum salt.
 20. The process of claim 19, wherein the admixingcomprises: a first step of admixing (i) the aluminium salt and (ii)histidine, to give a histidine/aluminium salt admixture; and a secondstep of admixing (i) said histidine/aluminium salt admixture and (ii)one or more antigens.
 21. The process of claim 20, wherein the antigeniccomposition is combined with another antigenic composition.
 22. Avaccine comprising the composition of claim 1 and a pharmaceuticallyacceptable carrier or excipient.
 23. The composition of claim 1comprising from about 2.5 mM to about 5 mM of free phosphate.
 24. Thecomposition of claim 7, wherein the aluminium salt is aluminumoxyhydroxide, aluminum hydroxyphosphate, or a mixture of two or more ofsaid salts.
 25. The composition of claim 10, wherein the sodium salt issodium phosphate.
 26. A composition comprising a nucleic acid encoding aprotein antigen, an aluminium salt and histidine, said compositionfurther comprising at least about 2.5 mM of free phosphate, wherein saidcomposition has a histidine to free phosphate molar ratio of at leastabout 1.25:1.
 27. A method for raising an immune response in a mammal,the method comprising administering to said mammal a compositioncomprising a mixture of antigens, essentially a single aluminium salt,and histidine, wherein said single aluminum salt is present in a ratioof at least 100:1 relative to any other aluminum salt in thecomposition.
 28. The method of claim 27, wherein the single aluminumsalt is an aluminum hydroxide or an aluminum phosphate.
 29. The methodof claim 28 wherein the single aluminum salt is aluminum oxyhydroxide oraluminum hydroxyphosphate.
 30. The method of claim 27, wherein thehistidine has a concentration between 1 mM and 10 mM.